Cell observation chamber

ABSTRACT

A biological cell observing chamber ( 30 ), wherein an intermediate support body ( 32 ) forming a skeleton outer shell structural part is mounted on a bottom support body ( 31 ) and a cover block body ( 33 ) is mounted on the bottom support body ( 31 ) on which the intermediate support body ( 32 ) is mounted by using lever mechanisms ( 36 ) and ( 37 ) incorporating a cam mechanism and a clamp mechanism so that the contact faces thereof can be pressed against each other in the vertical direction. The cam mechanism comprises cam grooves ( 36   b ) and ( 37   b ) formed in both leg part inner surfaces of U-shaped levers ( 36 ) and ( 37 ) rotatably supported on the bottom support body ( 31 ) and pins ( 40 ) and ( 41 ) slidably moving in the cam grooves and built in the outer peripheral surfaces of the intermediate support body ( 32 ) and the cover block body ( 33 ) at corresponding two points. Thus, in the biological cell observing chamber used for the detection of cell chemotaxis and chemotactic cell separator, the assembly and disassembly operations for the intermediate support body forming the skeleton outer shell structural part and the cover block body can be facilitated, and the operability of the chamber can be increased.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a cell observation chamber, andspecifically to a cell observation chamber comprising a plurality ofwells for keeping cells, a flow path for cells, and a window forobserving the movement of cells to function as the heart of an apparatusused for: for example, determining whether or not cells move in acertain direction by themselves; observing a state where cells move in acertain direction by themselves; measuring the number of cells that havemoved in a certain direction by themselves; and isolating cells thatmove in a certain direction by themselves, that is, an apparatus fordetecting cell chemotaxis and for isolating chemotactic cells.

2. Description of the Prior Art

There have conventionally been proposed and marketed various kinds ofapparatuses for detecting cell chemotaxis and for isolating chemotacticcells. In particular, there has been proposed an apparatus, as describedin Japanese Patent Laid-Open Publication No. 2002-159287, adapted to becapable of observing and quantitating the self-based movement of cellsprecisely and easily using a few cell samples to detect the chemotaxisof the cells due to chemotactic factor or the chemotaxis inhibition ofthe cells due to chemotactic factor inhibitor. In this apparatus, it isalso possible to isolate the cells utilizing the chemotaxis of thecells.

In the apparatus for detecting cell chemotaxis and for isolatingchemotactic cells described in the foregoing publication, a cellobservation chamber is arranged as follows.

As shown in FIG. 16, the cell observation chamber 00 comprises: acircular shallow dish-shaped bottom support body 01 with a window 01 cfor observing the movement of cells provided in the center of the bottompart thereof; a glass substrate 08 adapted to be placed on the bottompart 01 a of the bottom support body 01; a dish-shaped intermediatesupport body 02 adapted to be attached to the bottom support body 01 topress and fix the glass substrate 08 from above onto the bottom part 01a by connecting a cover 04 to be described hereinafter to the bottomsupport body 01 with screws; a substrate 07 and a packing member 010adapted to be fitted into a rectangular opening portion 02 c that isformed in the center of the bottom part of the intermediate support body02 to be fixed onto the glass substrate 08; a block body 09 adapted tobe fitted into the central recessed portion of the intermediate supportbody 02 to press and fix the substrate 07 onto the glass substrate 08through the packing member 010 using pressing screws not shown in thefigure; and a cover 04 adapted to be attached to the bottom support body01 through a screw connection to press and fix the block body 09 fromabove. The substrate 07 is made of silicon single-crystal material.

The connection between the bottom support body 01 and the intermediatesupport body 02 is to be made by screwing a male thread 02 d formed inthe outer peripheral surface of the body part of the intermediatesupport body 02 into a female thread 01 d formed in the inner peripheralsurface of the body part of the bottom support body 01 and by a screwconnection between the bottom support body 01 and the cover 04. Thescrew connection between the bottom support body 01 and the cover 04 isto be made by screwing a male thread 01 e formed in the outer peripheralsurface of the bottom support body 01 into a female thread 04 a formedin the inner peripheral surface of the sleeve part of the cover 04. Theintermediate support body 02 is to be positioned on the bottom supportbody 01 by inserting guide pins (not shown in the figure) disposed onthe upper surface of the body part of the bottom support body 01 intoguide pin receiving holes 02 f formed in the lower surface of the flangepart 02 b of the intermediate support body 02. Also, the block body 09is to be positioned in the intermediate support body 02 by insertingguide pins 013 disposed on the bottom surface of the intermediatesupport body 02 into guide pin receiving holes 09 a formed in the bottomsurface of the block body 09.

Then, in a state where the above components are assembled integrally andused, at least a pair of wells and a flow path for communicating ofthese wells are to be formed between the substrate 07 and the glasssubstrate 08. One of these wells is to be provided with cell suspension,while the other thereof is to be provided with chemotactic factorcontaining solution, so that cells move from one to the other of thewells through the flow path in response to the chemotactic factor. Amicroscopic observation is to be carried out through the window 01 c toobserve the state and to measure the number of moving cells.

The injection of cell suspension and chemotactic factor containingsolution into one and the other wells that are formed between thesubstrate 07 and the glass substrate 08 is to be performed using amicropipette through specialized through holes formed, respectively, inthe block body 09, the packing member 010, and the substrate 07. Afterassembling the bottom support body 01, the intermediate support body 02,and the cover 04, an O-ring 011 is to be interposed between theintermediate support body 02 and the glass substrate 08 so that nosolution filling the bottom support body 01 leaks. On the other hand,the packing member 010 is also provided between the substrate 07 and theblock body 09 so as to be useful in preventing each solution fromleaking from the wells and the flow path for communicating of the wells.

Since the conventional cell observation chamber is arranged as mentionedabove, the attachment of the intermediate support body 02 to the bottomsupport body 01 and of the cover 04 to the bottom support body 01 areboth achieved through a screw connection, which therefore requires aconstant pressure to bring the components into pressurized contact witheach other and thereby requires a specialized assembling tool, resultingin poor operationality and complication in disassembling and assembling.

Patent Document 1: Japanese Patent Laid-Open Publication No. 2002-159287

Patent Document 2: Japanese Patent Laid-Open Publication No. 2003-088357

SUMMARY OF THE INVENTION

The present invention has been made to solve the above-describedproblems of the conventional cell observation chamber, and an objectthereof is to provide a cell observation chamber that requires nospecialized assembling tool for the attachment of an intermediatesupport body to a bottom support body and of a block body integratedwith a cover to the bottom support body with the intermediate supportbody attached thereto, and adapted to be capable of keeping a constantpressure at any time to bring the associated components into pressurizedcontact with each other, resulting in a dramatic improvement inoperationality in assembling and disassembling.

In accordance with the present invention, the foregoing object can beachieved with the following cell observation chamber.

That is, the cell observation chamber is provided in an apparatus usedfor detecting cell chemotaxis and for isolating chemotactic cells, thechamber comprising: a dish-shaped bottom support body with a window forobserving the movement of cells provided in the center of the bottompart thereof; a glass substrate adapted to be placed on the bottomsurface of the bottom support body; a dish-shaped intermediate supportbody with an opening portion formed in the center of the bottom partthereof, the intermediate support body being adapted to be attached tothe bottom support body to press and fix the glass substrate from aboveonto the bottom surface of the bottom support body; a substrate with aplurality of through holes for guiding cell suspension and chemotacticfactor containing solution therethrough formed therein in a verticallypenetrating manner, the substrate being adapted to be fixed onto thesurface in the central part of the glass substrate, in which aconcavo-convex shape is formed in the surface facing the glass substrateto form at least a pair of wells and a flow path for communicating ofthe wells with the glass substrate; a packing member with a plurality ofthrough holes for guiding the cell suspension and the chemotactic factorcontaining solution therethrough formed therein in a verticallypenetrating manner, the packing member being adapted to be fitted intothe opening portion that is formed in the center of the bottom part ofthe intermediate support body to press the substrate from above; and adish-shaped cover block body with a plurality of through holes forguiding the cell suspension and the chemotactic factor containingsolution therethrough formed in the center of the bottom part thereof ina vertically penetrating manner, the cover block body being adapted tobe attached to the bottom support body with the intermediate supportbody attached thereto to press and fix the substrate from above onto theglass substrate through the packing member, wherein one of the pair ofwells is adapted to be provided or given with the cell suspensionthrough each one of the plurality of through holes that are formed,respectively, in the cover block body, the packing member, and thesubstrate, while the other of the wells is adapted to be provided orgiven with the chemotactic factor containing solution through each oneof the plurality of through holes that are formed, respectively, in thecover block body, the packing member, and the substrate, so that a statewhere cells move from one to the other of the wells through the flowpath is observed and the number of the cells is measured through thewindow provided in the bottom support body, and wherein the attachmentof the intermediate support body to the bottom support body and of thecover block body to the bottom support body is achieved by bringing therespective contact surfaces into vertically pressurized contact witheach other using lever mechanisms or clamp mechanisms with a cammechanism incorporated therein.

In accordance with the cell observation chamber, the attachment of theintermediate support body to the bottom support body and of the coverblock body to the bottom support body is achieved by bringing therespective contact surfaces into vertically pressurized contact witheach other using (a pair of) lever mechanisms or clamp mechanisms withthe cam mechanism incorporated therein, which eliminates the need for aspecialized tool for assembling and disassembling, whereby it ispossible to improve the operationality in assembling and disassemblingdramatically. This also makes it possible to keep a constant pressure atany time to bring the components associated with the attachment intopressurized contact with each other, whereby it is possible to preventthe solution in the chamber from leaking reliably.

In a preferred embodiment, the cam mechanism comprises: cam groovesformed, respectively, in both leg parts of two U-shaped levers that aresupported rotatably by the bottom support body; and pins implanted,respectively, at two corresponding points on the outer peripheralsurface of the intermediate support body and the cover block body, thepins being adapted to move within the cam grooves in a sliding manner.This allows the structure of the cam mechanism to be simplifiedsignificantly, and the attachment/detachment of the intermediate supportbody to/from the bottom support body and of the cover block body to/fromthe bottom support body can be achieved only by rotating the respectiveU-shaped levers, which facilitates the operation of the cam mechanismsignificantly.

In another preferred embodiment, a guide block body is further attachedto the cover block body, in the guide block body being formed aplurality of through holes for guiding a micropipette that has inhaledeither the cell suspension or the chemotactic factor containing solutiontherethrough in a vertically penetrating manner. This facilitatessignificantly the operation of injecting or removing the cell suspensioninto/from one of the wells in the cell observation chamber using themicropipette and the operation of injecting or removing the chemotacticfactor containing solution into/from the other of the wells in the cellobservation chamber using the micropipette.

As described heretofore, in accordance with the present invention, theattachment of the intermediate support body to the bottom support bodyand of the cover block body to the bottom support body is achieved bybringing the respective contact surfaces into vertically pressurizedcontact with each other using the lever mechanisms or the clampmechanisms with the cam mechanism incorporated therein, which eliminatesthe need for a specialized tool for assembling and disassembling,whereby it is possible to improve the operationality in assembling anddisassembling dramatically. This also makes it possible to keep aconstant pressure at any time to bring the components associated withthe attachment into pressurized contact with each other, whereby it ispossible to prevent the solution in the chamber from leaking reliably.

Also, in the case of further attaching the guide block body with aplurality of through holes for guiding the micropipette therethroughformed therein in a penetrating manner to the cover block body, theoperation of injecting or removing cell suspension and chemotacticfactor containing solution into/from the wells using the micropipettecan be facilitated significantly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram, partially in cross-section, of anembodiment of the cell observation chamber of the present inventionillustrating the flow path of cells between the wells of the chamber.

FIG. 2 is a top plan view of the cell observation chamber of FIG. 1.

FIG. 3 is a partial, enlarged schematic view, partially incross-section, of the cell observation chamber of FIG. 1 showing thecell flow path.

FIG. 4 is a bottom plan view of the substrate 7 of the cell observationchamber of FIG. 1.

FIG. 5 is a side, cross-sectional view of the substrate 7 of the cellobservation chamber of FIG. 1.

FIG. 6 is a perspective view of an apparatus for detecting cellchemotaxis and for isolating chemotactic cells, utilizing a cellobservation chamber of the present invention.

FIG. 7 is a perspective view of the cell observation chamber of FIG. 1.

FIG. 8 is a top plan view of the cell observation chamber of FIG. 1.

FIG. 9 is a front elevational view of the cell observation chamber ofFIG. 1.

FIG. 10 is a side elevational view of the cell observation chamber ofFIG. 1.

FIG. 11 is a front cross-sectional view of the cell observation chamberof FIG. 1.

FIG. 12 is a side cross-sectional view of the cell observation chamberof FIG. 1.

FIG. 13 is a perspective view of the cell observation chamber of FIG. 1,partially disassembled by removal of the guide block body 34.

FIG. 14 is a perspective view of the cell observation chamber of FIG. 1,further disassembled by removal of the cover block body 33.

FIG. 15 is a block diagram of a temperature control system for the cellobservation chamber of FIG. 1.

FIGS. 16(1) and 16(2) are exploded views of a conventional cellobservation chamber.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is arranged that the attachment of an intermediate support body to abottom support body and of a cover block body to the bottom support bodyfor constituting a cell observation chamber is achieved by bringing therespective contact surfaces into vertically pressurized contact witheach other using lever mechanisms or clamp mechanisms with a cammechanism incorporated therein. In this case, it is arranged that thecam mechanism comprises: cam grooves formed, respectively, in both legparts of two U-shaped levers that are supported rotatably by the bottomsupport body; and pins implanted, respectively, at two correspondingpoints on the outer peripheral surface of the intermediate support bodyand the cover block body, the pins being adapted to move within the camgrooves in a sliding manner, and that there are provided specialized cammechanisms, respectively, for the attachment of the intermediate supportbody to the bottom support body and of the cover block body to thebottom support body. It is also preferable that a guide block body formaking it easy to get the needlepoint of a micropipette in and out beattached to the cover block body.

Next will be described an embodiment of the present invention.

The principle of the operation of a chamber in an apparatus fordetecting cell chemotaxis and for isolating chemotactic cells to whichthe present invention is applied will first be described. In thischamber, a plurality of wells are connected and communicate with eachother through a flow path, in each well being provided two pipes: one isfor injecting or removing samples, and the other is for preventing thepressure in the well from increasing or decreasing due to the operationof injecting or removing the samples. These pipes may be formed bythrough holes formed in a block. It is here noted that the flow path isa part for communicating of two wells, that is, a channel through whichcells pass when moving from one to the other of the wells. In accordancewith the apparatus, since the liquid flow toward the opposite wells inthe flow path is unlikely to occur when injecting or removing samples,there is no possibility that the liquid in the wells provided on bothends of the flow path intermingles with each other, whereby it ispossible to detect the case where cells mainly move based only on theeffect of chemotactic factor.

To describe the principle based on the accompanying drawings, in FIGS. 1and 2, the numeral 1 indicates a flow path, and the numeral 2 indicateswells for storing samples such as cell suspension and specimen solution,consisting of a pair of wells 2A and 2B. These samples are provided orremoved to/from the wells 2 through the through holes 3 formed in theblock body 9 using a micropipette, etc. When one well 2A of the wells 2is provided with the cell suspension, cells try to move toward the otherwell 2B and pass through the flow path 1 if the specimen solution put inthe well 2B contains chemotactic factor (chemotactic factor containingsolution).

When providing the cell suspension, one sample, to the well 2A throughthe through hole 3 using a micropipette, etc., there is a possibilitythat cells move toward the well 2B provided on the opposite side throughthe flow path 1 due to the pressure of the liquid injected. Thissituation, if occurred, causes confusion about determining whether ornot the movement of the cells is due to the chemotactic factor containedin the specimen, and in the case of aiming at isolating cells, causesdesired cells to intermingle with other cells, which makes it impossibleto achieve the purpose. In order to solve the problem, this apparatus isarranged in such a manner that an injection pressure to be applied tothe through hole 3 is released toward the through hole 4 to preventcells from being flowed forcibly toward the flow path 1.

Also, when providing the specimen solution to the well 2B through thethrough hole 3 using a micropipette, etc., there is a possibility thatthe specimen solution enters the well 2A provided on the opposite sidethrough the flow path 1 due to the pressure of the liquid injected tointermingle with the cell suspension, and therefore the phenomenon thatcells pass through the flow path 1 due to the chemotaxis thereof may beconfused or disturbed. In order to prevent such a situation fromoccurring, a through hole 4 is also provided in the well 2B for storingthe specimen.

Thus providing the through holes 4 that communicate with the throughholes 3 for injecting the samples therethrough can minimize thehorizontal impact of the liquid pressure and thereby can determinewhether or not the specimen solution has chemotaxis more precisely. Theeffect of reducing pressure difference using the through holes 4 is alsoeffective in reducing pressure reduction when removing samples such ascells from the wells, which therefore makes it easy to remove thesamples.

To describe the case of injecting samples into the wells 2 in thisapparatus with reference to FIG. 1, the wells 2A and 2B and the flowpath 1 are preliminarily filled with cell isotonic solution, and thenapproximately the same quantity of cell suspension and chemotacticfactor containing solution is injected, respectively, through thethrough hole 3 of the well 2A and the through hole 3 of the well 2B.This allows the pressure increase when injecting the samples to bereduced by the through holes 4.

As shown in FIGS. 3 to 5, the flow path 1 is composed of one or aplurality of, for example about 100, grooves 5 formed in a barrier 6along the direction or the opposite direction from the well 2A towardthe well 2B, the barrier 6 running in the direction perpendicular to thedirection or the opposite direction from the well 2A toward the well 2B.These grooves 5 are formed at a width in accordance with the diameter ofcells or the deformability thereof. Thus providing the grooves 5 makesit possible to observe the cells at individual level and also to isolatethe cells into desired classes.

It is noted that the numeral 7 a in FIGS. 1 to 3 indicates a bank formedbetween the wells 2A and 2B, while the numeral 7 b in FIGS. 3 and 4indicates a terrace formed on the bank 7 a. The terrace 7 b is a flatportion surrounding the barrier 6.

An observation of a state where cells move through the flow path 1 and ameasurement of the number of cells that currently pass or have passedthrough the flow path 1 are to be made by setting a sensing device, forexample, a microscope 70 in such a manner that the sensing device facesthe flow path 1 through a glass substrate 8 as shown in FIG. 3. Also,combining the microscope with a video camera or a CCD camera makes itpossible to record the progress of the cell movement automatically.

Defining such an apparatus as mentioned above, in which the wells 2A and2B with the through holes 3 and 4 provided respectively therein arecommunicated with each other through the flow path 1, as one unit andintegrating a plurality of units makes it possible to construct anapparatus whereby the movement (chemotaxis) of cells can be detected andchemotactic cells can be isolated at the same time for other kinds ofspecimens or other kinds of cells. Since the size of such an apparatusis wholly reduced, it is possible to treat samples at a small quantity.In addition, the treatment can be automated easily with a programcontrol system for the injection/removal quantity of the liquid.

Such a unit as mentioned above, in which the wells 2A and 2B with thethrough holes 3 and 4 provided respectively therein are communicatedwith each other through the flow path 1, is actually manufactured asfollows.

The inner shape of the wells 2A and 2B and the flow path 1 can be formedby applying a known technique for manufacturing an integrated circuitonto the surface of a substrate 7 made of silicon single-crystalmaterial. Arranging the substrate 7 with a concavo-convex shape obtainedby thus transferring the inner shape of the wells 2A and 2B and the flowpath 1 thereto engraved on the surface thereof to face and overlap theglass substrate 8 causes the wells 2A and 2B and the flow path 1 to beformed between the substrates 7 and 8.

In the substrate 7, through holes 3′ for guiding cell suspension orchemotactic factor containing solution therethrough are also formedcorrespondingly to the respective wells 2A and 2B in a verticallypenetrating manner, and through holes 4′ for reducing pressure increaseor pressure reduction that occurs when injecting or removing thesolutions into/from the wells 2A and 2B are formed in pairs with therespective through holes 3′ in a vertically penetrating manner. Thesepairs of through holes 3′ and 4′ are communicated with each otherthrough the well 2A or 2B and communicate with the respective throughholes 3 and 4 that are formed in the block body 9 in a verticallypenetrating manner. It is noted that there is actually interposed apacking between the substrate 7 and the block body 9 so as to seal theliquid therebetween.

Next will be described in detail the cell observation chamber accordingto the present embodiment in which a plurality of such units asmentioned above, in which the wells 2A and 2B with the through holes 3and 4 provided respectively therein are communicated with each otherthrough the flow path 1, are incorporated.

The outline of the overall structure of the apparatus for detecting cellchemotaxis and for isolating chemotactic cells to which the cellobservation chamber according to the present embodiment is applied willfirst be described.

As shown in FIG. 6, in the apparatus 10 for detecting cell chemotaxisand for isolating chemotactic cells to which the cell observationchamber 30 according to the present embodiment is applied, the cellobservation chamber 30 is housed in such a manner as to be partiallyexposed on the upper surface of a relatively low casing 20 having arectangular parallelepiped shape. Also, a laptop computer 50 is placedon the upper surface of the casing 20, and the laptop computer 50 isadapted to operate to, for example, give instructions to a temperaturecontrol section for solutions containing cell suspension, etc. andanalyze, record, and monitor temperature data and/or cell observationdata. The monitoring includes displaying an image of an actual cellmovement.

Since a level 21 is additionally attached to the upper surface of thecasing 20, it is possible to monitor the evenness of the apparatus 10constantly. Further, a brightness (light intensity) adjustment knob 22for a cell observation image in the microscope, a position adjustmentknob 23 for the microscope, and a focal point adjustment lever 24, etc.are attached to the front surface of the casing 20 in this order fromthe lower right to the upper left in FIG. 6. Since an optical axis, notshown in the figure, in the optical system of the microscope is arrangedhorizontally in the casing 20, it is possible to reduce the height ofthe casing 20 and therefore the apparatus 10, which makes it possible toperform the operation of detecting cell chemotaxis, isolatingchemotactic cells, and measuring the number of cells in a sittingposture using the apparatus 10 placed on a desk, resulting in asignificant improvement in operationality.

The cell observation chamber 30 is arranged as follows.

As shown in FIGS. 7 to 10, 13, and 14, the arrangement of the cellobservation chamber 30 will be understood as follows based on theappearance thereof and a partially disassembled state achieved by asimple rotational operation of cam control levers 36 and 37 to bedescribed hereinafter. That is, onto a circular dish-shaped bottomsupport body 31 that is arranged in the lowest part is attached anintermediate support body 32 having also a circular dish shape; onto theintermediate support body 32 is attached a cover block body 33 havingalso a circular dish shape with the relatively thick bottom part 33 aand the relatively wide outer peripheral flange part 33 b; onto thecover block body 33 is attached a guide block body 34 across the centralrecessed portion 33 c of the cover block body 33 in such a manner thatthe central enlarged portion 34 a thereof is sunk into the centralrecessed portion 33 c; and on the upper surface of the cover block body33 is seated a pedestal part 35 a of a temperature sensor 35.

Then, rotating the cam control lever 36 causes the cover block body 33to be brought into pressurized contact with the intermediate supportbody 32 from above, which causes the intermediate support body 32 to bebrought into pressurized contact with the bottom support body 31 fromabove, so that the cover block body 33 is finally to be attached to thebottom support body 31. Also, rotating the cam control lever 37 causesthe intermediate support body 32 to be brought into pressurized contactwith the bottom support body 31 from above and to be attached thereto.It is noted that in an actual attachment order, the intermediate supportbody 32 is first attached to the bottom support body 31, and the coverblock body 33 is then attached to the bottom support body 31. In thecase of a disassembling operation, the operation is to be performed inthe reverse order. The cover block body 33 corresponds to one obtainedby combining the block body 09 and the cover 04 in the conventional cellobservation chamber 00 (refer to FIG. 16).

The cam control levers 36 and 37 each have a U shape when viewed fromabove, and the end portions 36 a and 37 a of the both leg parts thereofexist on the outer peripheral surface of the body part 31 b of thecircular dish-shaped bottom support body 31 to be supported rotatablyaround a pair of support shafts 38 that are implanted symmetrically withrespect to the axial center of the body part. Also, the end portions 36a and 37 a of the both leg parts are enlarged into a rectangular shapewhen viewed from front, in the inner surface of which being formedcurved cam grooves 36 b and 37 b, respectively, for the cam controllevers 36 and 37 (refer to FIGS. 13 and 14).

On the outer peripheral surface of the outer peripheral flange part 33 bof the circular dish-shaped cover block body 33, there are implantedpins 40 symmetrically with respect to the axial center of the flangepart (refer to FIGS. 14 and 11). The pins 40 are fitted into the camgrooves 36 b of the cam control lever 36 so as to move within the camgrooves 36 b in a sliding manner when the cam control lever 36 isrotated. This causes the lower surface of the outer peripheral flangepart 33 b of the cover block body 33 to come close to and to be broughtinto contact with the upper surface of the outer peripheral flange part32 b of the intermediate support body 32 from above, and finally to beattached to the bottom support body 31 while pressing a packing member44 and the substrate 7 that are fitted into an opening portion 32 c ofthe intermediate support body 32 against the glass substrate 8. Also,rotating the cam control lever 36 reversely causes the cover block body33 to be detached from the bottom support body 31. Between the outerperipheral flange part 33 b of the cover block body 33 and the outerperipheral flange part 32 b of the intermediate support body 32, thereis interposed an O-ring 42 for preventing medium from leaking from aninner space to be formed between the cover block body 33 and theintermediate support body 32 when the cover block body 33 is attached tothe intermediate support body 32.

Similarly, on the outer peripheral surface of the outer peripheralflange part 32 b of the circular dish-shaped intermediate support body32, there are implanted pins 41 symmetrically with respect to the axialcenter of the flange part (refer to FIG. 11). The pins 41 are fittedinto the cam grooves 37 b of the cam control lever 37 so as to movewithin the cam grooves 37 b in a sliding manner when the cam controllever 37 is rotated. This causes the lower surface of the outerperipheral flange part 32 b of the intermediate support body 32 to comeclose to and to be brought into contact with the upper surface of thebody part 31 b of the bottom support body 31 from above, and to beattached to the bottom support body 31 firmly. Also, rotating the camcontrol lever 37 reversely causes the intermediate support body 32 to bedetached from the bottom support body 31.

In the central enlarged portion 34 a of the guide block body 34, thereare formed six narrow through holes 34 c in a vertically penetratingmanner aligned in the longitudinal direction of the guide block body 34.When an operator inserts or withdraws the needlepoint of a micropipette(not shown in the figure) carrying a sample such as cell suspension orspecimen solution into/from the chamber 30, these through holes 34 c areuseful in guiding the needlepoint of the micropipette and in guiding thesolution discharged from the micropipette to the well to be describedhereinafter (this well is identical with one of the foregoing pair ofwells 2A and 2B (FIG. 1)). The position where the six through holes 34 care aligned is displaced slightly toward one side of a centerline “a”dividing the guide block body 34 into two sections in the widthdirection when viewed from above (refer to FIG. 8).

The guide block body 34 is positioned and attached onto the flange part33 b detachably with pins 39 penetrating through arm parts 34 b and 34 bon either side of the central enlarged portion 34 a and the flange part33 b of the cover block body 33. Therefore, after the guide block body34 is detached from the cover block body 33 and rotated by 180 degreesso that the positions of the arm parts 34 b and 34 b on either side areswitched with each other, the guide block body 34 can be attached againonto the flange part 33 b of the cover block body 33 detachably by beingpositioned using the pins 39 similarly before the switching. In thiscase, the position where the six through holes 34 c are aligned issymmetrical to the alignment position before the switching with respectto the centerline “a”.

In order to position the cover block body 33 and the intermediatesupport body 32 relatively in the circumferential direction, a pair ofpositioning pins 46 a and 46 b penetrate through holes formedrespectively therefor across the cover block body 33 and theintermediate support body 32. Similarly, in order to position theintermediate support body 32 and the bottom support body 31 relativelyin the circumferential direction, a pair of positioning pins 47 a and 47b penetrate through holes formed respectively therefor across theintermediate support body 32 and the bottom support body 31. The pins 46a and 46 b and the pins 47 a and 47 b have their respective differentdiameters to fulfill a function of preventing an assembling error in anassembling operation from occurring.

Next will be described the internal structure of the cell observationchamber 30 in detail.

In the center of the bottom part 31 a of the bottom support body 31,there is provided a window 31 c for observing the movement of cells.Also, the transparent glass substrate 8 is placed on the bottom surfaceof the body. When the intermediate support body 32 is attached to thebottom support body 31, the glass substrate 8 is pressed firmly againstand fixed to the bottom part 31 a by the bottom part 32 a of theintermediate support body 32. Between the bottom part 32 a and the glasssubstrate 8 and on the outer peripheral side thereof, there isinterposed an O-ring 43 to prevent medium from leaking from an innerspace to be formed therebetween.

The substrate 7 is placed on the surface in the central part of theglass substrate 8. The glass substrate 8 and the substrate 7 areidentical with the foregoing glass substrate 8 and substrate 7 in FIG. 1having basically the same structure. Therefore, on the surface of thesubstrate 7 facing the glass substrate 8, there are engraved six unitsof concavo-convex shapes obtained by transferring the inner shape of thepair of wells 2A and 2B and the flow path 1 for communicating of thewells thereto, and in a state where the shapes are arranged to face andoverlap the glass substrate 8, six units of combination structures ofthe wells 2A and 2B and the flow path 1 are formed between thesubstrates 7 and 8.

In the substrate 7, the through holes 3′ for guiding cell suspension orchemotactic factor containing solution therethrough are also formedcorrespondingly to the respective wells 2A and 2B in a verticallypenetrating manner, and the through holes 4′ for reducing pressureincrease or pressure reduction that occurs when injecting or removingthe solutions into/from the wells 2A and 2B are formed in pairs with therespective through holes 3′ in a vertically penetrating manner. Thesepairs of through holes 3′ and 4′ are communicated with each otherthrough the well 2A or 2B.

The opening portion 32 c is formed in the central part of the bottompart 32 a of the intermediate support body 32, and the packing member 44having a thickness slightly greater than that of the bottom part 32 a isfitted into the opening portion 32 c. When the cover block body 33 isattached to the bottom support body 31 as mentioned above, the packingmember 44 protrudes from the opening portion 32 c to press the substrate7 placed on the glass substrate 8 from above against the glass substrate8.

The substrate 7, which has a very small thickness, is represented as aheavy solid line segment sandwiched between the glass substrate 8 andthe packing member 44 in FIGS. 11 and 12. The shape of the through holes3′ and 4′, the wells 2A and 2B, and the flow path 1 formed in thesubstrate 7 is not shown in the figures.

In the packing member 44, there are formed the same number of throughholes 3-1 and 4-1 that communicate, respectively, with the through holes3′ and 4′ formed in the substrate 7 in a penetrating manner as the totalnumber of the through holes 3′ and 4′ in a vertically penetratingmanner. Since the through holes 3′ and 4′ are formed in each of thewells 2A and 2B in a pair, a total of four through holes are to beformed in one unit, and integrating six units causes a total of 24through holes (groups of through holes 3-1 and 4-1) to be formed andaligned lengthwise and crosswise. The through holes 3-1, which existdeeply and on the near side in the direction perpendicular to the spacein FIG. 11, are not shown in the figure.

It is noted that the through holes 3-1 and 4-1 to be formed in thepacking member 44 in a penetrating manner are not necessarily formedseparately, and the through holes 3-1 may be combined with therespective through holes 4-1. This cannot cause, for example, fallingsolution and rising gas to be intermingled with each other, and sincethe gas passes through the falling solution to be discharged through athrough hole 4-2 above, there is no interference with the function ofreducing pressure increase in the wells. In FIG. 12 is shown thestructure of thus arranged packing member 44. Also, for that purpose, ifthe lower end portions of through holes 3-2 and 4-2 to be formed in thecover block body 33 are cut off by a small length to form small blankspaces therein, it is possible to retain the function of reducingpressure increase and pressure reduction further reliably (refer to thetwo left and right small blank spaces directly below the through holes3-2 and 4-2 in FIG. 12).

When the cover block body 33 is attached to the bottom support body 31,the lower surface of the bottom part 33 a of the cover block body 33 isbrought into contact with the upper surface of the packing member 44 andpresses the surface. Therefore, the substrate 7 is consequently to bepressed by the cover block body 33 through the packing member 44 to befixed onto the glass substrate 8.

In one part nearer the peripheral edge of the bottom part 33 a of thecover block body 33, there is formed a relatively large-diameter throughhole 33 d in a vertically penetrating manner through which mixture inthe chamber 30 is adapted to go in and out of the central recessedportion 33 c. Also, in the central part of the bottom part 33 a, thereare formed the same number of through holes 3-2 and 4-2 thatcommunicate, respectively, with the through holes 3-1 and 4-1 formed inthe packing member 44 in a penetrating manner as the total number of thethrough holes 3-1 and 4-1 in a vertically penetrating manner. Amongthese groups of through holes formed in the central part of the bottompart 33 a, six units of the through holes 4-2 belonging to the well 2Aside, that is, six aligned through holes 4-2 belonging to the well 2Aside correspond one-on-one to the six through holes 34 c in the guideblock body 34 that is attached to the cover block body 33 in the postureas shown in FIG. 8 to share the centerline thereof.

When the guide block body 34 is rotated by 180 degrees from the postureas shown in FIG. 8 to switch the positions of the arm parts 34 b and 34b on either side with each other, six aligned through holes 4-2belonging to the well 2B side then correspond one-on-one to the sixthrough holes 34 c in the guide block body 34. Thus switching theposture of the guide block body 34 can be employed when the injection ofthe cell suspension into the well 2A using a micropipette is followed bythe injection of the chemotactic factor containing solution into thewell 2B using a micropipette.

As is clear from the description above, the through holes 3′ and 4′formed in the substrate 7 in a penetrating manner, the through holes 3-1and 4-1 formed in the packing member 44 in a penetrating manner, and thethrough holes 3-2 and 4-2 formed in the bottom part 33 a of the coverblock body 33 in a penetrating manner are communicated with each other,and six units of through hole assemblies that are formed by the throughholes 4′, 4-1, and 4-2 thus communicating with each other correspondone-on-one to the six through holes 34 c formed in the guide block body34 that is attached to the cover block body 33 in the posture as shownin FIG. 8 to share the centerline thereof (refer to FIGS. 11 and 12). Itis noted that the through holes 3′ and 4′ formed in the substrate 7 in apenetrating manner, which have very small sizes, are not shown in FIGS.11 and 12. The through hole assemblies composed of the through holes 4-1and 4-2 correspond to the through holes 4 in FIG. 1.

Accordingly, assuming here that the wells 2A and 2B and the flow path 1are filled with cell isotonic solution and that the well 2B is providedwith chemotactic factor containing solution, when trying to inject cellsuspension into the well 2A using a micropipette, after the needlepointof the micropipette is inserted into one of the through holes 34 c thatcommunicates with the well 2A in a unit to be used and is carried whilebeing guided by the hole until reaching a required depth to dischargethe cell suspension there, the discharged cell suspension then fallsdown through the through holes 4-2, 4-1, and 4′ in this order to reachthe well 2A. In this case, the pressure increase in the well 2A can bereleased outside through the through holes 3′, 3-1, and 3-2, which canminimize the impact of pressure fluctuation on the chemotaxis of cellsthat are to react with the chemotactic factor containing solution.

The same procedure applies also when trying to inject the chemotacticfactor containing solution into the well 2B using a micropipette, and inthis case, the chemotactic factor containing solution discharged fromthe micropipette can fall down through the through holes 4-2, 4-1, and4′ belonging to the well 2B side in this order to reach the well 2B.

Cells in the cell suspension provided to the well 2A move from the well2A to 2B through the flow path 1 after reacting with the chemotacticfactor containing solution in the well 2B. It is possible to observe thestate and measure the number of cells at the cell level through thewindow 31 c using the microscope.

In order to thus perform the operation of, for example, detecting thechemotaxis of the cells that move from the well 2A to 2B through theflow path 1 and isolating the cells utilizing the characteristicsthereof, it is necessary to control the temperature of the mixturefiling these sections so as to be suitable for the activity of thecells. Also when it is demanded that the reaction of the cells due totemperature change be measured and analyzed more precisely, it isnecessary to control the temperature of the mixture. It is noted thatthe mixture filling these sections here means the mixture of the cellisotonic solution and the cell suspension and the mixture of the cellisotonic solution and the chemotactic factor containing solution, wherethe both mixture has approximately the same temperature.

For the foregoing purpose, the present embodiment employs twotemperature controllers 62 and 63 as shown in FIG. 15, where the firsttemperature controller 62 is adapted to use the temperature sensor 35 tomeasure the temperature of the mixture directly and to control thetemperature of a heating section 64 to be heated by a heater with thechamber 30 being set thereon to increase the accuracy of the temperaturecontrol. Also, the second temperature controller 63 is adapted to heatthe heating section 64 preliminarily so that the time required tocontrol the temperature of the mixture to be a required temperature canbe shortened. The temperature controller 63 also has a function ofpreventing the heating section 64 from being overheated.

In order to measure the temperature of the mixture directly using thetemperature sensor 35, a temperature sensing part 35 b of thetemperature sensor 35 extends downward from the pedestal part 35 a, asshown in FIG. 12, to be directly sunk into a liquid storage chamber 45filled with solution equivalent to the mixture. The solution in theliquid storage chamber 45 can receive the indirect heating by theheating section 64 equally with the solutions filling the pair of wells2A and 2B and the flow path 1 to increase the temperature to be the sameas that of the solutions, whereby the temperature sensor 35 can measureapproximately the same temperature as that of the solutions filing thepair of wells 2A and 2B and the flow path 1. The liquid level of thesolution in the liquid storage chamber 45 is approximately the same asthe liquid level L of the mixture in the cover block body 33.

The liquid storage chamber 45 is formed with a recessed portion formedby partially and vertically chipping the outer peripheral wall of thebody part of the cover block body 33 being surrounded by the innerperipheral wall of the intermediate support body 32. It is preferablethat the liquid storage chamber 45 be provided separately from the wells2A and 2B, the flow path 1, and the sections communicating with theseportions. For this reason, it is arranged that there is interposed apacking (not shown in the figures) at the lower part of the liquidstorage chamber 45 where the liquid storage chamber 45 is connected tothe wells 2A and 2B, the flow path 1, and the sections communicatingwith these portions. This allows the temperature sensing part 35 b ofthe first temperature controller 62 to measure the temperature of thesolutions containing cells and filling the pair of wells 2A and 2B andthe flow path 1 precisely without contaminating the solutions.

To describe the in-chamber mixture temperature control system 60 in moredetail with reference to the block diagram shown in FIG. 15, when atemperature control switch 66 is first turned ON and a changeover switch67 is turned ON for preheating, a preheating operation for the heatingsection 64 is started under the control of the temperature controller63. The preheating operation is to be performed while measuring thetemperature of the heating section 64 using a sensor 65 and feeding backthe measured value. The preheating temperature is to be specified by acomputer 61. The computer 61 is incorporated in the laptop computer 50.The numeral 69 indicates a solid-state relay (SSR).

When the temperature of the heating section 64 reaches a predeterminedpreheating temperature and the cell observation chamber 30 is placed onthe heating section 64, the changeover switch 67 is turned ON forheating to start a heating operation for the heating section 64 underthe control of the temperature controller 62. This heating operation,which is aiming at heating the mixture in the chamber to be apredetermined temperature, is to be performed while measuring thetemperature of the mixture in the chamber using the sensor 35 andfeeding back the measured value. The heating temperature is to bespecified by the computer 61. Since the heating section 64 has beenheated to be the predetermined temperature through the foregoingpreheating operation, this heating operation can heat the mixture in thechamber to be the predetermined temperature in a short time.

When the temperature of the mixture in the chamber reaches thepredetermined temperature, the temperature controller 62 performsheating control for the heating section 64 to keep the temperature. Ifthe temperature of the heating section 64 increases abnormally (e.g. 43°C.) for some reasons, for example, that the chamber 30 is not in contactwith the heating section 64, the temperature controller 63 operates therelay 68 to shut off the circuit. It is noted that the temperaturecontroller 62 is also adapted to operate the relay 68 to shut off thecircuit if the temperature of the mixture in the chamber increasesabnormally (e.g. 38 to 40° C.).

The computer 61 is adapted to monitor and display the temperature of theheating section 64 and the mixture in the chamber, the state of thesensors 35 and 65, etc. constantly, and to specify a heating temperatureand a preheating temperature, respectively, for the temperaturecontrollers 62 and 63.

Here will be described in detail an actual procedure for assembling thecell observation chamber 30 according to the present embodiment.

The glass substrate 8 is first attached to the bottom support body 31.Then, the intermediate support body 32 is fitted into the bottom supportbody 31, and the cam control lever 37 is rotated to bring theintermediate support body 32 into pressurized contact with the bottomsupport body 31 from above through the O-ring 43 and to attach theintermediate support body 32 to the bottom support body 31. This canprevent medium from leaking to give the assembly composed of thesecomponents a function as a container. Next, the substrate 7 is placed onthe glass substrate 8 while being guided by the opening portion 32 cformed in the central part of the bottom part 32 a of the intermediatesupport body 32, and the cover block body 33 with the packing member 44attached to the bottom surface thereof is fitted into the intermediatesupport body 32, and then the cam control lever 36 is rotated to bringthe packing member 44 into pressurized contact with the substrate 7 fromabove and to bring the substrate 7 into pressurized contact with theglass substrate 8. At the same time, the cover block body 33 is broughtinto pressurized contact with the intermediate support body 32 throughthe O-ring 42 so that it is possible to prevent the medium from leakingto give the general assembly (cell observation chamber 30) composed ofthese components also a function as a container.

The cell observation chamber 30 according to the present embodiment,which is thus arranged, can exhibit the following effects.

The attachment of the intermediate support body 32 to the bottom supportbody 31 and of the cover block body 33 to the bottom support body 31 isachieved by bringing the respective contact surfaces into verticallypressurized contact with each other using the lever mechanisms (camcontrol levers) 36 and 37 with the cam mechanism incorporated therein,which eliminates the need for a specialized tool for assembling anddisassembling, whereby it is possible to improve the operationality inassembling and disassembling dramatically. This also makes it possibleto keep a constant pressure at any time to bring the components (bottomsupport body 31, intermediate support body 32, and cover block body 33)associated with the attachment into pressurized contact with each other,whereby it is possible to prevent the solutions in the chamber fromleaking reliably.

Further, it is arranged that the cam mechanism comprises: the camgrooves 36 b and 37 b formed, respectively, in the both leg parts of thetwo U-shaped levers 36 and 37 that are supported rotatably by the bottomsupport body 31; and the pins 40 and 41 implanted, respectively, at twocorresponding points on the outer peripheral surface of the cover blockbody 33 and the intermediate support body 32, the pins being adapted tomove within the cam grooves 36 b and 37 b in a sliding manner. Thisallows the structure of the cam mechanism to be simplifiedsignificantly, and the attachment/detachment of the intermediate supportbody 32 to/from the bottom support body 31 and of the cover block body33 to/from the intermediate support body 32 can be achieved only byrotating the respective U-shaped levers 36 and 37, which facilitates theoperation of the cam mechanism significantly.

In addition, the guide block body 34 is further attached to the coverblock body 33, in the guide block body 34 being formed a plurality ofthrough holes 34 c for guiding the micropipette that has inhaled eitherthe cell suspension or the chemotactic factor containing solutionstherethrough in a vertically penetrating manner. This facilitatessignificantly the operation of injecting or removing the cell suspensionand the chemotactic factor containing solution into/from the wells inthe cell observation chamber 30 using the micropipette.

It is possible to additionally exhibit various kinds of such effects asmentioned above.

It is noted that the present invention is not restricted to theabove-described embodiment, and various modifications may be madewithout departing from the gist thereof.

For example, instead of lever mechanisms, such as the cam control levers36 and 37, with a cam mechanism incorporated therein, it is possible toemploy a clamp mechanism based on a toggle clamp system or a compressionclamp system in which a nail is adapted to clamp after compression.

1. A cell observation chamber for use in an apparatus for detecting cellchemotaxis and for isolating chemotactic cells, said chamber comprising:a dish-shaped bottom support body with a window for observing themovement of cells provided in the center of a bottom surface thereof; aglass substrate adapted to be placed on the bottom surface of saidbottom support body; a dish-shaped intermediate support body with anopening in the center of a bottom part thereof, said intermediatesupport body being adapted to be attached to said bottom support body topress and fix said glass substrate from above onto the bottom surface ofsaid bottom support body; a substrate having, in a surface facing saidglass substrate, at least a pair of wells and a flow path providingcommunication between said wells, said substrate further having aplurality of through holes for guiding cell suspension and chemotacticfactor containing solution therethrough and into said wells, saidsubstrate being adapted to be fixed onto the surface in the central partof said glass substrate; a packing member with a plurality of throughholes for guiding said cell suspension and said chemotactic factorcontaining solution therethrough formed therein, said packing memberbeing adapted to be fitted into said opening in the center of the bottompart of said intermediate support body to press said substrate fromabove; and a dish-shaped cover block body with a plurality of throughholes for guiding said cell suspension and said chemotactic factorcontaining solution therethrough formed in the center of a bottomthereof, said cover block body being adapted to be attached to saidbottom support body with said intermediate support body attached theretoto press and fix said substrate from above onto said glass substratethrough said packing member; a first cam mechanism comprising a pair ofpins symmetrically arranged on and extending from a periphery of saidcover block body and a first U-shaped cam lever pivotally mounted onsaid bottom support body and including a first handle and a first pairof legs respectively fixed to opposing ends of the handle, each of saidfirst legs having a cam groove receiving one of the pins extending fromsaid cover block body, whereby movement of said first cam lever from afirst position to a second position forces together said cover blockbody and said bottom support body, with said intermediate support bodyclamped therebetween; and a second cam mechanism comprising a pair ofpins symmetrically arranged on and extending from a periphery of saidintermediate support body and a second U-shaped cam lever pivotallymounted on said bottom support body and including a second handle and asecond pair of legs respectively fixed to opposing ends of the secondhandle, each of said second legs having a cam groove receiving one ofthe pins extending from said intermediate support body, whereby movementof said second cam lever from a first position to a second positionforces together the intermediate support body and said bottom supportbody.
 2. The cell observation chamber according to claim 1, furthercomprising: a guide block body attached to said cover block body, saidguide block body having a plurality of through holes for guiding amicropipette to dispense either said cell suspension or said chemotacticfactor containing solution therethrough into one of said wells.
 3. Thecell observation chamber according to claim 1, further comprising: apair of support shaft portions symmetrically arranged on and extendingfrom said bottom support body; and wherein said legs of said first andsecond cam levers are mounted on said. support shaft portions.
 4. Thecell observation chamber according to claim 1, wherein: said cover blockbody and said intermediate support body each have a peripheral flange;and said bottom support body has a rim presenting an annular surface;and further comprising: first holes in a surface of the peripheralflange of the cover block body and second holes aligned with the firstholes and formed in a surface of the peripheral flange of theintermediate support body facing the flange of the cover block body anda first pin received in each of said second holes and in a first holealigned therewith for circumferentially positioning said cover blockbody on said intermediate support body; and third holes in anothersurface of the peripheral flange of the intermediate support body facingthe bottom support body and fourth holes aligned with the third holesand formed in the annular surface of said bottom support body and asecond pin received in each of said third holes and in a fourth holealigned therewith for circumferentially positioning said intermediatesupport body on said bottom support body.
 5. The cell observationchamber according to claim 4, wherein said first pins have differentdiameters and said second pins have different diameters.